新生鼠反复低血糖引起神经元变性的动态观察

目的 建立新生鼠低血糖脑损伤模型，并观察损伤后不同脑区神经元变性的动态变化。方法 160只新生Wistar大鼠随机分为胰岛素诱导短时间低血糖组(INS-S组，n=40)、胰岛素诱导长时间低血糖组(INS-P组，n=40)、饥饿组(FAS组，n=40)和对照组(CON组，n=40)。在大鼠出生后第2、4、6天分别采用皮下注射胰岛素(15U/kg)和饥饿的手段，诱导新生鼠低血糖；在生后第6天诱导低血糖后2h、6h、1d、3d、7d和14d采用Fluoro-Jade B(FJB)染色法观察7个脑区变性神经元的动态变化，并应用特异性神经核抗原(NeuN)荧光免疫双标对FJB阳性细胞进行细胞类型鉴定。结果 INS-S组、FAS组和CON组新生大鼠各脑区各时间点均未见FJB阳性细胞，而INS-P组在旁矢状面、梨状皮层、海马齿状回、丘脑和下丘脑可见大量的FJB阳性细胞，并同时显示NeuN免疫源性。结论 胰岛素(15U/kg)皮下注射可建立稳定可靠的新生鼠低血糖脑损伤模型。新生鼠反复严重低血糖可引起广泛的神经元变性，皮层、丘脑、下丘脑和海马齿状回对低血糖损害更为敏感。

Neurodegeneration in different brain regions of neonatal rats damaged by hypoglycemia

Objective   To establish a reliable animal model of brain injury induced by neonatal hypoglycemia and observe the  corresponding neurodegeneration in different brain regions of neonatal rats. Methods    Newborn Wistar rats were randomly divided into: insulin-treated rats with short hypoglycemia (INS-S group, n=40), insulin treated rats with prolonged hypoglycemia (INS-P group, n=40), fasting rats (FAS group, n=40) or control rats (CON group, n=40). Neonatal hypoglycemia was produced by fasting or insulin injections (15U/kg, hypodermic injection) on postnatal day 2 (P2), 4 (P4), and 6 (P6). Double labeled immunofluorescent with neuronal nuclear protein (NeuN) and FJB was performed to examine the neuronal degeneration in the rat brain 2h, 6h, 1d, 3d, 7d and 14d after the third hypoglycemic insult. Results   No FJB+ cells were detected in INS-S, FAS and CON rats at any designated time point, but numerous FJB+ cells were detected in INS P rats at 6h, 1d, 3d and 7d after the third hypoglycemic insult. Double labeled immunofluorescent with NeuN showed these FJB+ cells all presented NeuN immunoreactivity. There were numerous FJB+ cells in the parasagittal cortex, piriform cortex, hypothalamus, thalamus and dentate gyrus. Conclusion    Insulin hypodermic injections in newborn rat can establish a reliable animal model of brain injury damaged by neonatal hypoglycemia. Repetitive and profound neonatal hypoglycemia can result in extensive neurodegeneration, in which the neurons of the cortex, thalamus, hypothalamus and dentate gyrus are more vulnerable.